Periplasmic Toxoid Extraction via pH Shock and Osmotic Release
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Solution Overview
Problem
The production of significant quantities of diphtheria toxins like CRM197 for vaccines is hindered by low protein abundance and challenges in expressing recombinant fusion proteins in E. coli, leading to degraded proteins and decreased host cell viability due to periplasmic secretion.
Innovation Solution
A process involving maturing the host cell through pH shock, incubation without feed addition, or freezing below -20°C, followed by osmotic shock extraction, to enhance the efficiency of protein extraction and yield in periplasmic expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If periplasmic expression is used to produce bacterial toxoid, then protein yield is improved, but host cell viability decreases and proteolysis increases
Solution Approach 1:
The patent applies preliminary action by adding a maturation step before protein extraction. This step includes incubating the host cell under specific conditions (pH shock, temperature changes, or nutrient depletion) to optimize the periplasmic environment prior to extraction, thereby improving protein yield while maintaining host cell viability and reducing proteolysis.
2Productivity
If periplasmic expression is used to produce bacterial toxoid, then protein yield is improved, but proteolysis increases
Solution Approach 1:
The maturation step is performed before extraction to prepare the periplasmic environment in advance. This preliminary action optimizes conditions to minimize proteolytic activity during storage and extraction, thereby protecting the expressed protein from degradation while maintaining high yield.
Solution Approach 2:
The patent employs parameter changes by modifying pH, temperature, and nutrient conditions during the maturation step. These parameter changes create an environment that suppresses proteolytic activity while maintaining protein stability and solubility in the periplasm, thus reducing harmful proteolysis.
3Productivity
If extraction efficiency is increased through optimized osmotic shock, then protein yield is improved, but process complexity increases
Solution Approach 1:
The maturation step serves as a preliminary action that simplifies the subsequent extraction process. By optimizing the periplasmic environment before extraction, the osmotic shock step becomes more efficient and requires fewer additional steps or complex equipment, thereby improving extraction efficiency without significantly increasing process complexity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This process significantly increases the efficiency of protein extraction and yield, improving the production of recombinant bacterial toxoids like CRM197, addressing issues of viability and proteolysis associated with periplasmic expression.
Implementation Method 1
subjecting the host cell to a pH shock
Implementation Method 2
freezing the host cell to a temperature below -20°C
Implementation Method 3
extracting the bacterial toxoid from the host cell wherein the extraction process comprises osmotic shock
Data Source
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AI summary
The present invention provides a process for periplasmic expression of a bacterial toxoid comprising the steps of: a) growing a culture of a gram negative host cell in a fermentation medium, wherein the host cell is transformed with a polynucleotide, and wherein the polynucleotide encodes the bacterial toxoid and a periplasmic signal sequence; or providing a gram negative host cell wherein the host cell is transformed with a polynucleotide, the polynucleotide encodes the bacterial toxoid and a periplasmic signal sequence and wherein the gram negative host cell comprises the bacterial toxoid expressed in the periplasm; a(i)) inducing expression of the bacterial toxoid; b) maturing the host cell, wherein the maturing step comprises: I) subjecting the host cell to a pH shock; II) incubating the host cell with no feed addition; and/or III) subjecting the host cell to a temperature below -20°C; and c) extracting the bacterial toxoid from the host cell wherein the extraction process comprises osmotic shock.